Aging is associated with more than a 10-fold increase in the incidence of sudden cardiac death (SCD). Cellular senescence is a stress response that is characterized by an irreversible loss of proliferative capacity, accompanied by a complex senescence-associated secretory phenotype (SASP) that can have profound effects on neighboring cells in the tissue. Our research plan is based on the following main premises. First, that fibrosis is a consequence of heart injury (myocardial infarction) by being part of the natural wound healing response, and that inflammatory processes in the scar may predispose the heart to arrhythmias. Second, that cellular senescence of cardiac fibroblasts (CFs) plays an important role in post-MI wound healing. The overarching hypothesis of our proposal is that senescence is an important component of wound healing in the heart. Specifically, we postulate that in the aging human heart with a healed MI, the interplay between aging cardiomyocytes (CMs) that have a lower threshold for arrhythmogenic activity, and the a modified senescence response observed in the aging heart may lead to arrhythmogenesis. Furthermore, we hypothesize that aging also impacts the senescence of cardiac fibroblasts (CFs) by either affecting the kinetics of the senescence response, the intrinsic properties of the senescent cells, or their clearance by the immune system. This multi-PI proposal brings together several investigators with very different expertise to investigate mechanisms of SCD in the aging heart, using a novel age-appropriate large animal model (rabbit), CFs-specific genetically modified mice, and engineered 3D microtissues of CMs and CFs where their interactions can be studied in vitro. In Aim 1 the Koren group will investigate the infarct healing process in the aging rabbit heart, as compared to the young heart, and correlate the arrhythmogenesis with molecular and cellular analysis of CMs and CFs and myofibroblasts derived from the IBZ and infarct zone (IZ) as compared to the remote zone (RZ). In Aim 2 the Sedivy group will use mouse models to focus on the role CFs senescence in this process. Senescence will be manipulated in vivo using genetic and pharmacologic approaches, CFs will be cultured and investigated in vitro. Optical mapping of hearts ex vivo will be used to study arrhythmogenesis. In Aim 3 the Mende and Choi groups will study the effect of senescent CFs on excitation, conduction and calcium handling in biomimetic cardiac microtissues, in which CMs and senescent CFs can interact via direct cell-cell contact. In summary, we envision that by understanding the aging mechanisms that control cellular senescence we will be able to reduce fibrosis and the incidence of SCD.